246 USE OF IONOPHORE A23187 ON BOVINE FROZEN SPERM INCREASES HYPERACTIVATED MOTILITY

2015 ◽  
Vol 27 (1) ◽  
pp. 212
Author(s):  
R. Garaguso ◽  
M. J. Franco ◽  
N. M. Ortega ◽  
T. Fanti ◽  
A. A. Mutto
Keyword(s):  
Tyrosine Phosphorylation ◽  
Female Reproductive Tract ◽  
Ionophore A23187 ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Control Groups ◽  
Protein Tyrosine ◽  
Motility Pattern ◽  
Phosphorylation Pattern ◽  
Bovine Spermatozoa

Sperm capacitation is critical for oocyte fertilization in mammals. The capacitated state is acquired by the spermatozoon during its passage through the female reproductive tract and can be induced in vitro. At a functional level, sperm capacitation is associated with changes in the motility pattern – the hyperactivated state (HA) – and terminates with the acrosome reaction (AR). These events are characteristically regulated by different Ca2+-signalling pathways. For this reason, Ca2+ ionophores, such as A23187, are commonly used in sperm capacitation studies. The induction of AR and IVF, as well as the assessment of protein phosporylation of tyrosine substrates are useful methods for the evaluation of the capacitation state of spermatozoa. Although the increase of protein tyrosine phosphorylation via PKA is associated with sperm capacitation, in the mouse, A23187 capacitates the spermatozoa, thus bypassing this pathway. The aim of the present work was to test the effect of the ionophore A23187 on acquisition of the capacitated state by evaluation of HA motility and protein phosphorylation pattern in frozen bovine spermatozoa. Motile bovine spermatozoa were isolated by gradient centrifugation (Percoll) from frozen/thawed samples and treated with different concentrations of A23187 (0.05, 0.1, 0.2, and 0.3 µM) in H-TYH medium without BSA. After 10 min of treatment, spermatozoa were washed in medium with BSA (11.2%) and incubated in H-TYH with BSA (6%) for 30 min. For control groups, sperm were incubated in H-TYH medium and DMSO. The motility pattern was visually identified and quantified using a computer-assisted sperm analysis system. The motile/immotile spermatozoa and the HA motility patterns of each group were statistically analysed by applying Fisher's tests. The protein tyrosine phosphorylation pattern was evaluated by a Western immunoblotting assay using heparin as a positive control of sperm capacitation. Our results showed that spermatozoa treated with A23187 had a significant increase in HA motility. The proportions of HA spermatozoa were 10.92, 31.27, and 75.4% for 0.1, 0.2, and 0.3 µM A23187, respectively (P < 0.05). On the other hand, the pattern of PKA-tyrosine phoshorylation characteristic of capacitated spermatozoa was absent after incubation with A23187, similar to the response seen in mouse spermatozoa. The percentage of motile spermatozoa in the control groups (H-TYH medium: 36% and DMSO: 27.95%; P < 0.05) was reduced as compared with that of the basal sperm suspension (65.4%, P < 0.05) after 30 to 40 min of incubation. Motility in the spermatozoa treated with 0.1 and 0.2 µM ionophore was similar – 65.9 and 68.1%, respectively, but it was reduced in the 0.3 group – 57.5% (P < 0.05). Thus, treatment with A23187 increased the percentage of spermatozoa with HA motility, probably suggesting an improvement in fertilizing capacity. Nevertheless, these promising results remain to be confirmed by IVF assay.

Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation

Development ◽  
1995 ◽  
Vol 121 (4) ◽  
pp. 1129-1137 ◽  
Author(s):  
P.E. Visconti ◽  
J.L. Bailey ◽  
G.D. Moore ◽  
D. Pan ◽  
P. Olds-Clarke ◽  
...  
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Tyrosine Phosphorylation ◽  
Bovine Serum ◽  
Female Reproductive Tract ◽  
Dependent Manner ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Protein Tyrosine

The molecular basis of mammalian sperm capacitation, defined functionally as those processes that confer on the sperm the acquisition of fertilization-competence either in vivo in the female reproductive tract or in vitro, is poorly understood. We demonstrate here that capacitation of caudal epididymal mouse sperm in vitro is accompanied by a time-dependent increase in the protein tyrosine phosphorylation of a subset of proteins of M(r) 40,000-120,000. Incubation of sperm in media devoid of bovine serum albumin, CaCl2 or NaHCO3, components which individually are required for capacitation, prevent the sperm from undergoing capacitation as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. In each of these cases the protein tyrosine phosphorylation of the subset of capacitation-associated proteins does not occur. Protein tyrosine phosphorylation of these particular proteins, as well as sperm capacitation, can be recovered in media devoid of each of these three constituents (bovine serum albumin, CaCl2 or NaHCO3) by adding back the appropriate component in a concentration-dependent manner. The requirement of NaHCO3 for these phosphorylations is not due to an alkalinization of intracellular sperm pH or to an increase in media pH. Caput epididymal sperm, which lack the ability to undergo capacitation in vitro, do not display this capacitation-dependent subset of tyrosine phosphorylated proteins in complete media even after extended incubation periods, and do not fertilize metaphase II-arrested eggs in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Signal transduction mechanisms involved in in vitro ram sperm capacitation

Reproduction ◽  
2006 ◽  
Vol 132 (5) ◽  
pp. 721-732 ◽  
Author(s):  
Patricia Grasa ◽  
José Álvaro Cebrián-Pérez ◽  
Teresa Muiño-Blanco
Keyword(s):  
Tyrosine Phosphorylation ◽  
Calcium Influx ◽  
Calcium Entry ◽  
Calcium Entry Blocker ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Protein Tyrosine ◽  
Entry Blocker ◽  
Ram Sperm

We validate the chlortetracycline (CTC) technique for the evaluation of capacitation and acrosome reaction-like changes in ram sperm, carrying out a double estimation of the acrosome status after treatment with lysophosphatidylcholine, using fluorescein isocyanate (FITC)-RCA/ethidium homodimer 1 (EthD-1) and CTC/EthD-1. Highly consistent results and a positive correlation between the results of acrosome-reacted sperm evaluated with both techniques were obtained. In this study, we evaluate the effects of ram sperm capacitation of BSA, Ca2+, NaHCO3and cAMP agonists and their influence on the associated protein tyrosine phosphorylation. We found a time-dependent increase in capacitation related to protein tyrosine phosphorylation, either in the absence or the presence of BSA. The addition of an increasing concentration of cholesterol to samples containing BSA did not influence results. The effect of bicarbonate was concentration-dependent, with a significantly lowered value of non-capacitated sperm in the presence 18 and 25 mM. The addition of extracellular calcium did not significantly increase either the proportion of capacitated sperm or the protein tyrosine phosphorylation signalling, although a significantly higher value of acrosome-reacted sperm was found in samples containing 4 mM Ca2+. cAMP agonists increased capacitated sperm and protein tyrosine phosphorylation signalling. The inhibition of protein kinase A by H-89 caused a decrease in sperm capacitation. Addition of a calcium-entry blocker (Verapamil; Sigma) did not influence results, which suggests that the calcium entry blocker was unable to inhibit the calcium influx associated with capacitation in ram sperm. Our findings might benefit our understanding of the biochemical mechanisms involved in mammalian sperm capacitation and ultimately, fertility.

93 CALCIUM REMOVAL INCREASES THE PROTECTIVE EFFECTS OF β-CYCLODEXTRIN PLUS CHOLESTEROL ON PORCINE SPERM DURING COLD SHOCK

2006 ◽  
Vol 18 (2) ◽  
pp. 155 ◽  
Author(s):  
H. Galantino-Homer ◽  
W. Zeng ◽  
S. Megee ◽  
M. Modelski ◽  
I. Dobrinski
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cold Shock ◽  
Cholesterol Content ◽  
Extracellular Calcium ◽  
Shock Treatment ◽  
Membrane Cholesterol ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Viability ◽  
Sperm Capacitation ◽  
Protein Tyrosine

Porcine sperm are extremely sensitive to the damaging effects of cold shock and cryopreservation. Cholesterol-binding molecules, such as 2-hydroxypropyl-�-cyclodextrin (HBCD), improve post-thaw and post-cooling porcine sperm viability when added to an egg yolk-based extender, but also enhance sperm capacitation in other species. Depending upon the environmental cholesterol content, HBCD can act either as a cholesterol shuttle or sink to increase or decrease, respectively, sperm plasma membrane cholesterol content. Increasing the sperm cholesterol to phospholipid ratio reduces cold shock sensitivity whereas decreasing the ratio initiates the process of sperm capacitation. An increase in protein tyrosine phosphorylation correlates with sperm capacitation and has been shown to be dependent upon the presence of extracellular calcium. Sperm intracellular calcium also increases during cold shock. The objective of this study was to determine the combined effects of extracellular calcium and membrane cholesterol manipulation on porcine sperm viability and protein tyrosine phosphorylation following cold shock (10�C for 10 min). Viability was assessed using CFDA/propidium iodide staining. Protein tyrosine phosphorylation, previously shown to correlate with porcine sperm capacitation, was evaluated via antiphosphotyrosine (clone 4G10) immunoblots. We report here that following cold shock, porcine sperm incubated in defined medium containing both 0.8 mM HBCD and 0.5 mM cholesterol 3-sulfate (ChS) incubated in the absence of added extracellular calcium and the presence of 6 mM EGTA have significantly improved viability (90.5 � 6.3%, n = 3) when compared with cold-shocked sperm incubated in either the same medium with calcium (46.1 � 3.8%), without HBCD or ChS (26.5 � 7.4% with calcium; 46.5 � 13.1% without calcium), or with HBCD alone (17.0 � 7.4% with calcium, 36.8 � 7.5% without calcium). As we have found previously, treatment with 0.8 mM HBCD plus 0.5 mM ChS completely inhibited the increase in protein tyrosine phosphorylation induced by the cold shock treatment. Although protein tyrosine phosphorylation correlates with porcine sperm capacitation, the ability of cold shock treatment to induce the same phosphorylation pattern indicates that other processes or pathways may contribute to its appearance. Removing extracellular calcium consistently decreased, but did not completely eliminate, the protein tyrosine phosphorylation induced by cold shock. These results indicate that cold shock-induced protein tyrosine phosphorylation is not dependent upon, but can be modulated by, extracellular calcium. The combined effects of calcium, HBCD and ChS on viability suggest that porcine sperm viability following cold shock is best maintained by removing extracellular calcium and increasing membrane cholesterol content via the cholesterol shuttle activity of HBCD. This work was supported by grants from PA Dept. Ag. (ME 443291) and the NIH (5-K08-HD041430).

scholarly journals Nitric Oxide Regulates Human Sperm Capacitation and Protein-Tyrosine Phosphorylation In Vitro1

1999 ◽  
Vol 61 (3) ◽  
pp. 575-581 ◽  
Author(s):  
Maria Belén Herrero ◽  
Eve de Lamirande ◽  
Claude Gagnon
Keyword(s):  
Nitric Oxide ◽  
Tyrosine Phosphorylation ◽  
Human Sperm ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Protein Tyrosine

Luteinizing hormone modulates intracellular calcium, protein tyrosine phosphorylation and motility during human sperm capacitation

2017 ◽  
Vol 483 (2) ◽  
pp. 834-839 ◽  
Author(s):  
Aideé S. López-Torres ◽  
María E. González-González ◽  
Esperanza Mata-Martínez ◽  
Fernando Larrea ◽  
Claudia L. Treviño ◽  
...  
Keyword(s):  
Luteinizing Hormone ◽  
Intracellular Calcium ◽  
Tyrosine Phosphorylation ◽  
Human Sperm ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Protein Tyrosine

scholarly journals Cholecystokinin receptors regulate sperm protein tyrosine phosphorylation via uptake of HCO3−

Reproduction ◽  
2015 ◽  
Vol 150 (4) ◽  
pp. 257-268 ◽  
Author(s):  
Yuchuan Zhou ◽  
Yanfei Ru ◽  
Huijuan Shi ◽  
Yanjiao Wang ◽  
Bin Wu ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Human Sperm ◽  
Peptide Hormone ◽  
Mature Sperm ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Protein Tyrosine ◽  
Cck Receptors ◽  
Cck2 Receptor

Cholecystokinin (CCK), a peptide hormone and a neurotransmitter, was detected in mature sperm two decades ago. However, the exact role of CCK and the types of CCK receptors (now termed CCK1 and CCK2) in sperm have not been identified. Here, we find that CCK1 and CCK2 receptors are immunolocalized to the acrosomal region of mature sperm. The antagonist of CCK1 or CCK2 receptor strongly activated the soluble adenylyl cyclase/cAMP/protein kinase A signaling pathway that drives sperm capacitation-associated protein tyrosine phosphorylation in dose- and time-dependent manners. But these actions of stimulation were abolished when sperm were incubated in the medium in the absence of HCO3−. Further investigation demonstrated that the inhibitor of CCK1 or CCK2 receptor could accelerate the uptake of HCO3−and significantly elevate the intracellular pH of sperm. Interestingly, the synthetic octapeptide of CCK (CCK8) showed the same action and mechanism as antagonists of CCK receptors. Moreover, CCK8 and the antagonist of CCK1 or CCK2 receptor were also able to accelerate human sperm capacitation-associated protein tyrosine phosphorylation by stimulating the influx of HCO3−. Thus, the present results suggest that CCK and its receptors may regulate sperm capacitation-associated protein tyrosine phosphorylation by modulating the uptake of HCO3−.

scholarly journals Protein tyrosine phosphorylation in rabbit peritoneal neutrophils

1990 ◽  
Vol 269 (2) ◽  
pp. 431-436 ◽  
Author(s):  
C K Huang ◽  
V Bonak ◽  
G R Laramee ◽  
J E Casnellie
Keyword(s):  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Leukotriene B4 ◽  
Early Event ◽  
Ionophore A23187 ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Group A ◽  
Group B ◽  
Stimulation Of

Protein tyrosine phosphorylation in rabbit peritoneal neutrophils was examined by immunoblotting with antibodies specific for phosphotyrosine. Stimulation of the neutrophils with chemotactic factor fMet-Leu-Phe (10 nM) caused rapid increases of tyrosine phosphorylation of several proteins with apparent molecular masses of (Group A) 54-58 kDa and 100-125 kDa and (Group B) 36-41 kDa. Stimulation of Group A proteins was observed by fMet-Leu-Phe (10 nM, maximum at 20 s) and A23187 (1 microM, 1 min). Stimulation of Group B proteins was observed by fMet-Leu-Phe (ED50 0.15 nM, 1 min), leukotriene B4 (ED50 0.15 nM, 1 min), phorbol 12-myristate 13-acetate (PMA) (ED50 25 ng/ml, 10 min) and partially by ionophore A23187 (1 microM, 1 min). Pretreatment of the cell with the protein kinase inhibitor H-7 (25 microM, 5 min) and PMA (0.1 microgram/ml, 3 min) partially inhibited the fMet-Leu-Phe effect. However, pretreatment of the cells with quin 2/AM (20 microM, 10 min) completely inhibited the fMet-Leu-Phe effect. The results indicate that rapid regulation of tyrosine phosphorylation is an early event occurring in stimulated neutrophils. Furthermore the effect of fMet-Leu-Phe on tyrosine phosphorylation may require Ca2+ mobilization and may partially require the activity of H-7-sensitive protein kinases.

scholarly journals Intracellular calcium and protein tyrosine phosphorylation during the release of bovine sperm adhering to the fallopian tube epithelium in vitro

Reproduction ◽  
2005 ◽  
Vol 129 (1) ◽  
pp. 51-60 ◽  
Author(s):  
Roberto Gualtieri ◽  
Raffaele Boni ◽  
Elisabetta Tosti ◽  
Maria Zagami ◽  
Riccardo Talevi
Keyword(s):  
Tyrosine Phosphorylation ◽  
Ionophore A23187 ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Phosphorylated Proteins ◽  
Bovine Sperm ◽  
Surface Remodeling ◽  
Bovine Species

In mammals, sperm adhesion to the epithelial cells lining the oviductal isthmus plays a key role in the maintenance of motility and in the selection of superior quality subpopulations. In the bovine species, heparin and other sulfated glycoconjugates powerfully induce the synchronous release of sperm adhering to tubal epithelium in vitro and may represent the signal which triggers release at ovulation in vivo. Sperm detachment may be due either to surface remodeling or to hyperactivation brought about by capacitation. In this paper, the dynamics of intracellular free Ca2+concentration ([Ca2+]i) and protein tyrosine phosphorylation in sperm during and after heparin-induced release from in vitro cultured oviductal monolayers were assessed to determine whether this event is due to capacitation. Moreover, Ca2+-ionophore A23187, thapsigargin, thimerosal and caffeine were used to determine whether [Ca2+]i increase and/or hyperactivation can induce sperm release. Results showed that: 1. heparin-released sperm have significantly higher [Ca2+]i than adhering sperm; 2. heparin induces a [Ca2+]i elevation in the sperm head followed by detachment from the monolayers; 3. external Ca2+is not required for heparin-induced release; 4. [Ca2+]i increase and/or hyperactivation are unable to release sperm; and 5. heparin-released sperm have an increased level of tyrosine phosphorylated proteins compared with adhering sperm. In conclusion, although heparin is considered a long-lasting capacitation agent, it quickly modulates the capacitation of bovine sperm adhering to the fallopian epithelium, probably leading to surface remodeling and therefore to the release of sperm selected and stored within the oviduct through adhesion.

244 PROTEIN TYROSINE PHOSPHORYLATION PATTERN OF SPERM BOUND TO THE PORCINE OVIDUCT DURING THE PERIOVULATION STAGE

2013 ◽  
Vol 25 (1) ◽  
pp. 270
Author(s):  
V. Luño ◽  
R. López-Úbeda ◽  
L. Lefièvre ◽  
C. Matás
Keyword(s):  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphorylation ◽  
Fixed Tissue ◽  
Protein Tyrosine ◽  
Boar Sperm ◽  
Phosphorylation Pattern ◽  
Paraffin Block ◽  
Low Levels ◽  
High Level ◽  
One Way Anova

The interaction of spermatozoa and oviductal epithelial cells (OEC) is a controlled process that regulates sperm capacitation and the acquisition of fertilizing ability until the time of ovulation. A crucial signalling event involved in capacitation is protein tyrosine phosphorylation. In previous studies, we have demonstrated changes in the pattern of protein tyrosine phosphorylation in boar sperm after the co-culture with OEC. The aim of this study was to characterise the pattern of protein tyrosine phosphorylation in boar sperm bound or unbound to the oviduct of the sow during the periovulation stage. Eight crossbred multiparous sows were inseminated with 3 × 109 sperm. The animals were anesthetized and laparotomies were performed at 36 h after insemination. Ovaries and oviducts were exposed through a midventral incision for collection. Each oviduct was divided into four parts: the ampulla, ampullary-isthmic junction, isthmus, and utero-tubal junction. All segments of the oviduct were flushed to recover spermatozoa, which were subsequently fixed. Tissue obtained from each of the oviduct segments were fixed and embedded in a paraffin block. Sections were mounted on poly-l-lysine-coated slides and deparaffinized. Flushed sperm and oviductal sections were analysed by indirect immunofluorescence using monoclonal antiphosphotyrosine antibodies. Three different sperm subpopulations were determined according to the distribution of protein tyrosine phosphorylation observed: nonphosphorylated spermatozoa (pattern 1), subequatorial segment or subequatorial segment and flagellum phosphorylation (pattern 2), and subequatorial segment and head or flagellum phosphorylation, or both (pattern 3). Data were analysed with SPSS (IBM, Armonk, NY, USA) using one-way ANOVA. After flushing, most sperm were recovered from the utero-tubal junction segment of the oviduct, and sperm exhibited a higher proportion of pattern 2 (81.62%). Unbound sperm showed a high level of protein tyrosine phosphorylation in the subequatorial segment, head and flagellum in the isthmus (32.34%), ampullary-isthmic junction (37.70%), or ampulla region (35.11%; P < 0.05). Very few sperm were attached to OEC, and sperm oviduct binding was mainly found in the isthmus region. The most common tyrosine phosphorylation distribution observed in sperm attached to OEC was pattern 1 (84.21%), although labelling to the subequatorial segment was also observed. Our results showed that only sperm that did not display tyrosine phosphorylation on the sperm acrosome region (head) were found bound to OEC. In conclusion, distinct protein tyrosine phosphorylation patterns were found on sperm bound to OEC. This interaction could be used as a tool for selecting a population of sperm containing low levels of tyrosine phosphorylation.

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